Although the link between the immune system and the gut microbiome is being increasingly appreciated, the mechanistic links between the microbiome and inflammatory autoimmune diseases such as multiple sclerosis (MS) are unclear. Thus, there is a critical need to define mechanisms by which gut bacteria maintain immune homeostasis and thereby affect neuroinflammation. We have recently reported that MS patients have altered gut microbiota compared to healthy controls, with a reduced abundance of certain human gut bacteria (Parabacteroides, Adlercreutzia, and Prevotella) with the ability to metabolize phytoestrogen. Gut bacteria metabolize phytoestrogen into equol which has structural similarity with estrogen, and can regulate immune responses through estrogen receptors and AMP-activated protein kinase (AMPK). Our preliminary data showing significantly milder EAE in mice on a phytoestrogen (Isoflavones) containing diet compared to those on a phytoestrogen free diet suggest a critical role of phytoestrogen in modulation of EAE. Additionally, Prevotella histicola, a member of the Prevotella genus, can suppress disease in experimental autoimmune encephalomyelitis (EAE), a preclinical murine model of MS. However, the importance of phyoestrogen metabolism in P. histicola?mediated disease suppression is unknown. The central hypothesis of the proposed studies is that the human gut commensal P. histicola mediate its disease-protective effect through metabolism of phytoestrogen and subsequent activation of immunoregulatory cells. We will test our central hypothesis using animal model, genetically modified mice, and a cell culture approach in the following two specific aims. In first aim, we will utilize P. histicola as a representative phytoestrogen-metabolizing gut commensal bacteria to determine whether metabolism of phytoestrogen is required for ability of P. histicola to suppress disease and induce Tregs and/or CD103+ Tolerogenic dendritic cells (DCs). We will determine the importance of AMPK signaling in the phytoestrogen induced activation of Tregs and/or tolerogenic DCs. In second aim, we will determine whether the phytoestrogen plus bacteria mediate the induction of immunoregulatory cells via estrogen receptor (ER)-dependent pathways in intestinal epithelial cells and/or immune cells. Our study is fit for the ?High Priority Immunology Grant (R01) mechanism because we are proposing to determine the mechanisms underlying the microbial impact on systemic immunity which can be harnessed in designing potential mono as well as combination therapies because drugs with diverse non-overlapping mechanisms might provide maximal benefit to MS patients. We will determine whether phytoestrogen-metabolizing bacteria maintain a disease-free state by tilting the Tregs to Th1/Th17 balance towards Tregs through its interaction with estrogen receptors and the AMPK pathway. We expect the outcome of our study will have a positive impact on development of gut microbial-flora/diet-based therapies for MS as well as other inflammatory autoimmune diseases.